Some chemicals physically bind to rodent feed. This binding usually increases with time. This phenomenon may also have an effect on the absorption of the chemical after ingestion by rodents. The objective of these studies is to determine if there is a difference in absorption of study chemicals in rats given freshly prepared vs. aged chemical feed mixes. In an earlier NTP study the physical binding of o-nitroanisole (ONA) to NIH-07 rodent feed was found to increase with storage time. To evaluate this phenomenon, the systemic availability of ONA from aged fresh feed formulations was determined by following the urinary excretion of ONA metabolites in male F344 rats analytical methods for the determination of ONA in feed and its major metabolite, o-nitrophenol (free and conjugated), in rat urine were validated. The results of this investigation indicated that there was no difference in the systemic availability of o-nitroanisole from fresh or aged feed formulations and therefore the physical binding of ONA to NIH-07 rodent feed had no effect on the absorption of the chemical after ingestion by F344 rats.